This invention relates to the detection of variations in human H2 receptors, and more particularly to the development of new compounds useful in the sequencing and identification of a human histamine H2 receptor and their use in the diagnosis and treatment of certain human disorders, for example, brain disorders. The invention also relates to new compounds and a method for detecting an allelic polymorphic variation within the human population for the gene encoding the histamine H2 receptor and their use in the diagnosis and treatment of human disorders.
The human H2 receptor was first identified by Black et al Nature (1972), 236, 385-390. This was followed by the demonstration of the receptor in the mammalian brain by Baudry et al (1975) Nature 253, 362-363, and Haas and Bucher (1975) Nature 255, 634-635. Gantz et al (1991) Biochem. Biophys. Res. Comm. 178, 3, 1386-1392 have recently identified the sequence of a human H2 receptor cDNA from gastric parietal cells by using the polymerase chain reaction (PCR) and degenerated oligonucleotide primers whose sequence was obtained from the canine H2 receptor previously cloned by this group, Gantz et al (1991) Proc. Nat. Acad. Sci. USA 88, 429-433. This sequence was characterised as an intronless gene encoding a typical seven transmembrane domain aminergic receptor protein.
The receptor is coupled to heterotrimeric GTPases (G proteins), but differs from other monoamine receptors in this G protein coupled superfamily in several respects. The human gastric H2 receptor is shorter than most other. receptors in this class (359 amino acids) and lacks the two serine residues in the fifth transmembrane region (TM5). There exists instead an aspartate and a threonine residue, so far unique in this region. These two residues may be important for binding with the nitrogen atoms of the imidazole ring of histamine as suggested by Birdsall (1991) Trends in Pharmacological Sci. Jan, 12, 9-10.
Histamine is a natural constituent of many organs and tissues including the gastrointestinal tract, the immune system and the brain, Green et al (1987) Agents and Actions 22, 1-15. It is a central neurotransmitter in the brain and is formed in the posterior hypothalamus from exogenous histidine by histidine decarboxylase (HDC). It is subsequently metabolised by histamine methyltransferase (HMT), Prell et al (1986) Ann. Rev. Neurosci. 9, 209-254. The cell bodies and neuronal pathways for histamine have been mapped in the human brain using immunocytochemistry by Panula et al (1990) Neuroscience 34, 127-132. Its cells project from the tuberomamillary nucleus of the posterior hypothalamus to almost every region of the brain. There are three known histamine receptors; H1, H2 and H3, the latter functioning as an autoreceptor. The H2 receptor specifically has been localised in the human brain by Traiffort et al (1992) J. of Neurochem. 59, 1, 290-299. using receptor autoradiography.
Histamine is known to have significant effects in the central nervous system (CNS). It has been implicated in the CNS mediated mechanisms of arousal ever since the sedating effect of H1 receptor antagonists (eg,. chlorpheniramine, chloropromazine) had been noticed clinically. The use of H2 receptor antagonists in the human brain however, has shown, that these compounds, unlike those acting on the H1 receptor, do not produce any effect on psychomotor functioning, or a subjective feeling of sedation or arousal in healthy subjects, White et al (1988) Psychopharmacology 95, 1-14. Some H2 receptor antagonists (eg. cimetidine) are known to cause confusion in elderly or severely medically ill patients, perhaps in part due to a co-existing anti-cholinergic effect. H1 and H2 receptor antagonists in large doses have been reported to cause hallucinations, Csillag et al (1973) Med. J. Aug. 1, 653-654, Argawal (1978) J. Am. Med. Assoc. 240, 214. Animal studies have shown that histamine applied directly to the hippocampus, where there is the highest level of activity of the H2 receptor, will induce psychomotor withdrawal and decreased exploratory behaviour. The above evidence has led to the conclusion that H1 receptor systems are excitatory in the terms of arousal and motivated behaviour whilst H2 receptor systems are inhibitory in this respect, Alvarez and Banzan (1985) Physiol. and Beh. 34, 661-664 and (1986) Physiol. and Beh. 37, 39-45, White et al (1988) supra.
The H2 receptor is a site of action of various compounds used in the treatment of psychiatric disorders eg. amitriptyline and mianserin, Traiffort et al (1992) supra. Kaminsky et al (1990) The Lancet 335, 1351-1352 and (1991) Schizophrenia Bull. 4, 318-319 have reported the successful response of patients with chronic, predominantly negative type schizophrenia, to the highly specific H2 receptor antagonist famotidine. For example in one patient there was a substantial amelioration of the deficit symptoms of schizophrenia (eg apathy, social withdrawal, and blunted affect) while on famotidine, relapse in these symptoms on withdrawal, and improvement on re-institution of this drug, Kaminski, U.S. Pat. Nos. 5,070,101 and 5,177,081. Prell et al (1992) Abstract, part 1, 199.6 Soc. for Neurosci. Annual Meeting, Anaheim Cal. have shown substantially raised levels of N-tele-methyl histamine, a metabolite of histamine in the cerebrospinal fluid of patients with schizophrenia which correlates with those patients with the occurrence of negative symptoms of this disorder assessed using the Psychiatric Symptoms Assessment Scale. These levels were not significantly different between patients free from medication and those on neuroleptic therapy. It is therefore postulated that there is an increase in histaminergic activity in patients with chronic schizophrenia.
The disclosures of all the above mentioned publications are incorporated herein by reference for all purposes.
Additionally, histamine, acting via its receptors, including the H2 receptor, is believed to be critically involved in a number of diseases of organs other than the brain; these include peptic ulceration, allergic reactions, including asthma, immune-mediated disorders, and possibly some tumours.
The histamine H2 receptor is one of many receptors in the body. Compounds used to treat many diseases work by activating a receptor or inhibiting the action if its natural ligand. Variations in receptors amongst the population are known to be caused by allelic variation and this variation, can alter the response of a disease to a drug amongst patients. An example of this would be the response to clozapine, used to treat, schizophrenia associated with allelic variation in the 5-HT2A receptor demonstrated by Arranz el al (1995) Lancet, 346(8970), 291-282.
The present invention is concerned in one aspect with improvements in the diagnosis and/or treatment of human neurological and psychiatric disorders, and more particularly in the diagnosis and treatment of schizophrenia. In another aspect, the invention is concerned with improvements in the diagnosis and/or treatment of diseases of other systems or organs of the human body.
As a first step to the present invention, the Applicants devised a new oligonucleotide probe to the human H2 receptor mRNA in accordance with the published cDNA sequence available for the gastric parietal cell. Surprisingly, studies using this probe with in-situ hybridization histochemistry on human post-mortem brain tissue produced evidence of a mismatch in the nucleotide sequence for the brain H2 receptor and the sequence for the gastric parietal cell H2 receptor. This discovery was made by recording melt-curve estimations for the optimum hybridization incubation temperature using the method of Davis et al (1986) xe2x80x9cBasic Methods in Molecular Biologyxe2x80x9d page 77 Elsevier Science Publishing Co. It was found that the sequence mismatch is of the order of 10%.
It was apparent, therefore, that there is a hitherto unrecognised allele or subtype of the human histamine H2 receptor gene, which may be specific to the brain.
In a first aspect, the invention provides a sequence for a novel allele of a human histamine H2 receptor gene comprising up to six single base substitutions compared with the cDNA sequence published by Gantz et al (1991) Biochem Biophys Res Comm 178, 3, 1386-1392 as follows:
In another aspect, the invention provides a nucleotide sequence coding for a region of a human histamine H2 receptor, comprising one or more of the following base substitutions compared with the published sequence in Gantz et al (1991) supra, and from which the positional notation is taken:
The nucleotide sequence of the invention can, for example, comprise the following sequence (as also listed in SEQ ID NO:1):
As a specific exemplification, the nucleotide sequence of the invention can comprise the following sequence (as also listed in SEQ ID NO:2):
In another aspect of the invention, a series of new oligonucleotide primers have been developed for the identification of sequences in a sample comprising a human histamine H2 receptor DNA, cDNA or RNA originating from a tissue sample or body fluid.
In this aspect, the invention provides new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H2 receptor, having nucleotide sequences selected from:
1) 5xe2x80x2 CCAATGGCACAGCCTCTT 3xe2x80x2 (as listed in SEQ ID NO: 3)
2) 5xe2x80x2 CGTGACTTCTGTCCCACT 3xe2x80x2 (as listed in SEQ ID NO: 4)
3) 5xe2x80x2 CCAGGCAACAGGAAGAGA 3xe2x80x2 (as listed in SEQ ID NO: 5)
4) 5xe2x80x2 TCTCTTCCTGTTGCCTGG 3xe2x80x2 (as listed in SEQ ID NO: 6)
5) 5xe2x80x2 GCAGCAGAAGAGCTGTTG 3xe2x80x2 (as listed in SEQ ID NO: 7)
6) 5xe2x80x2 TCCAGGTCAATGAAGTGT 3xe2x80x2 (as listed in SEQ ID NO: 8)
7) 5xe2x80x2 ACACTTCATTGACCTGGA 3xe2x80x2 (as listed in SEQ ID NO: 9)
8) 5xe2x80x2 CCAAGAGGATCAATCACA 3xe2x80x2 (as listed in SEQ ID NO: 10)
9) 5xe2x80x2 TGTGATTGATCCTCTTGG 3xe2x80x2 (as listed in SEQ ID NO: 11)
and a diagnostic kit comprising one or more of the new oligonucleotides.
The direction and base start numbers for the novel oligonucleotide primers are as follows:
Information on the human histamine H2 receptor was obtained from the MRC Daresbury database accessing xe2x80x9cGenem 61xe2x80x9d File no. M64799xe2x80x94Human histamine H2 receptor gene.
The above mentioned substitutions alter and in some instances introduce or remove new sites for cleavage by specific restriction endonucleases as follows:
The invention further provides a diagnostic kit comprising one or more of the new oligonucleotide primers and preferably, one or more of the above mentioned endonucleases, optionally with one or more buffers.
A kit may be used to establish genotype or base variations. This information may be used in predicting an individuals disease susceptibility, disease course, prognosis and/or response to treatment as would be understood by those skilled in the art from the disclosure contained therein. The treatment response or efficacy which may be predicted may include drug treatment such as for example, use of the H2 receptor antagonist like famotidine or other forms of treatment such as social or psychological intervention.
Eucaryotic expression vectors comprising a DNA sequence coding for a protein and/or a peptide according to the invention are new materials and are also included in the invention. Host cells, for example, cloned human cell lines, such as NTera 2 c.dl, can be transformed using the new expression vectors and are also included in the invention.